The development of impaired renal function during sepsis predicts a poor outcome and increases the risk for mortality. The pathogenic mechanisms that underlie renal tubule dysfunction during sepsis are poorly understood and the identification of effective therapeutic approaches remains a priority. In preliminary studies, we demonstrate that absorption of HCO3- by the medullary thick ascending limb (MTAL) is inhibited by lipopolysaccharide (LPS). These studies provide the first evidence that bacterial molecules act directly through Toll-like receptors to impair the transport function of renal tubules, thereby identifying a new pathophysiological mechanism contributing to renal tubule dysfunction during bacterial infection. Monophosphoryl lipid A (MPLA) is a detoxified derivative of LPS that retains the parent compound's beneficial immunomodulatory activities without the proinflammatory side effects. Our preliminary studies show that treatment with MPLA attenuates kidney dysfunction in a mouse model of polymicrobial sepsis induced by cecal ligation and puncture (CLP) and prevents sepsis-induced impairment of MTAL HCO3- absorption. Accordingly, the Specific Aims are: AIM I. Identify the transport and signaling mechanisms through which basolateral LPS inhibits HCO3- absorption in the MTAL. These experiments will test the hypothesis that basolateral LPS decreases HCO3- absorption by inhibiting the apical NHE3 Na+/H+ exchanger through activation of the ERK signaling pathway, mediated through a novel interaction of Toll-like receptor 4 (TLR4) and TLR2. AIM II. Identify the transport and signaling mechanisms through which lumen LPS inhibits HCO3- absorption. These experiments will test the hypothesis that lumen LPS decreases HCO3- absorption by inhibiting basolateral NHE1 through activation of the PI3K-mTOR signaling pathway, mediated through TLR4. AIM III. Determine mechanisms by which sepsis decreases HCO3- absorption in the MTAL. These experiments will test the hypothesis that CLP sepsis impairs MTAL HCO3- absorption through a novel two hit mechanism, involving the combination of a decrease in baseline transport capacity and potentiation of inhibition by LPS. We propose that sepsis-induced transport inhibition is mediated through activation of ERK and results in increased urinary HCO3- excretion that contributes to sepsis-induced metabolic acidosis. AIM IV. Determine mechanisms by which MPLA protects against sepsis-induced MTAL transport inhibition. These experiments will test the hypothesis that MPLA stimulates the PI3K-Akt pathway through TLR4 and Trif, which prevents sepsis-induced inhibition of HCO3- absorption through downregulation of ERK. The studies proposed in this application will use a multidisciplinary approach to examine cellular and molecular mechanisms through which the bacterial molecule LPS impairs the transport function of the MTAL, the importance of these mechanisms in the pathogenesis of MTAL dysfunction during sepsis, and mechanisms through which the novel therapeutic agent MPLA protects against sepsis-induced alterations in MTAL function.